A 100% water-soluble surfactant polymer dressing (SPD) that is bio-compatible and non-ionic has been reported to improve wound closure in preliminary clinical studies. The mechanism of action of SPD in wound healing remains unclear. Biofilm infection is a significant problem that hinders proper wound closure. The objective of this study was to characterize the mechanism of action of SPD inhibition of bacterial biofilm development. Static biofilms (48 h) of the primary wound pathogens Pseudomonas aeruginosa (PA01), Staphylococcus aureus (USA300) were grown on polycarbonate membranes and treated with SPD with and without antibiotics for an additional 24 h. The standard antibiotics - tobramycin (10 μg/ml) for PA01 and rifampicin (10 μg/ml) for USA300, were used in these studies. Following 24 h treatment with and without antibiotics, the biofilms were characterized using scanning electron microscopy (SEM) structural imaging, in vitro imaging system (IVIS) proliferation imaging, colony forming units (CFU), viability assay, quantitative PCR (qPCR) for virulence gene expression. Because SPD is a surfactant based dressing, it potentially has a direct effect on Gram negative bacteria such as Pseudomonas primarily due to the lipid-based outer membrane of the bacteria. SPD is a surfactant based dressing that has potent anti-biofilm properties directly or in synergy with antibiotics.